Limits...
A putrescine-anthracene conjugate: a paradigm for selective drug delivery.

Palmer AJ, Ghani RA, Kaur N, Phanstiel O, Wallace HM - Biochem. J. (2009)

Bottom Line: Apoptosis was the predominant type of cell death, with mechanistic studies revealing that oxidative stress and DNA damage may have a part to play.For the first time, uptake of Ant 4 via the PTS was demonstrated both directly and indirectly in human cell lines.In addition, Ant 4 significantly reduced putrescine uptake, demonstrating that this conjugate not only used the PTS, but also could successfully compete with its native polyamine for uptake.

View Article: PubMed Central - PubMed

Affiliation: Divison of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen AB25 2ZD, UK.

ABSTRACT
Increased polyamine concentrations play an important role in the development of cancer at all stages, from initiation through to maintenance of the transformed phenotype. One way cancer cells accumulate increased concentrations of polyamines is by increased uptake of preformed polyamines via their PTS (polyamine transport system). The PTS is promiscuous and will transport a range of polyamine-based molecules. Therefore it may be that cytotoxic drugs could be attached to polyamine vectors and targeted selectively to cancer cells by utilizing the PTS. The aim of the present study was to investigate the potential of Ant 4, a putrescine-anthracene conjugate, to target cytotoxic agents to human cancer cells as a paradigm for a novel method of selective drug delivery. Ant 4 induced cytotoxicity after only 24 h exposure. Apoptosis was the predominant type of cell death, with mechanistic studies revealing that oxidative stress and DNA damage may have a part to play. For the first time, uptake of Ant 4 via the PTS was demonstrated both directly and indirectly in human cell lines. In addition, Ant 4 significantly reduced putrescine uptake, demonstrating that this conjugate not only used the PTS, but also could successfully compete with its native polyamine for uptake. However, the most interesting finding was the intracellular depletion of the polyamine pools, providing an additional mode of toxicity for Ant 4 and the possibility that this molecule may act as a 'double-edged sword': preventing cell growth by delivery of the toxic moiety and by depletion of intracellular polyamine content.

Show MeSH

Related in: MedlinePlus

HL-60 viable cell number in the presence of Ant 4HL-60 cells were seeded at 6.8×104 cells/ml in 3.5-cm plates and grown for up to 144 h. After 48 h, cells were treated with vehicle (■) or 15 μM Ant 4 (▲). Duplicate plates were harvested at 0 h and then every 24 h. Viable cell number and cell viability were determined by Trypan Blue exclusion and microscopy. Protein content was determined uising the Lowry assay. Results are the means±S.E.M. (n=4) with two replicates per experiment. Results were analysed statistically using ANOVA with Bonferroni's multiple comparison post-hoc test (*P<0.01; **P< 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2805923&req=5

Figure 2: HL-60 viable cell number in the presence of Ant 4HL-60 cells were seeded at 6.8×104 cells/ml in 3.5-cm plates and grown for up to 144 h. After 48 h, cells were treated with vehicle (■) or 15 μM Ant 4 (▲). Duplicate plates were harvested at 0 h and then every 24 h. Viable cell number and cell viability were determined by Trypan Blue exclusion and microscopy. Protein content was determined uising the Lowry assay. Results are the means±S.E.M. (n=4) with two replicates per experiment. Results were analysed statistically using ANOVA with Bonferroni's multiple comparison post-hoc test (*P<0.01; **P< 0.001).

Mentions: The IC50 value, at which 50% inhibition of cell growth was observed, was calculated to be 20 μM after 48 h exposure. Multiple studies from our laboratory have shown that IC50 values calculated from MTT curves give an overestimate of the value (F.R. Saunders and H.M. Wallace, unpublished work). Therefore in order to examine the response to Ant 4 over time in greater detail, a concentration of 15 μM was chosen (~IC30 value). At this concentration, inhibition of growth was observed by decreases in both cell number (Figure 2) and protein content (result not shown). Cytotoxic effects were observed after 24 h exposure to Ant 4. Despite the low cell numbers at longer times, cell viability remained high (86.5%), a feature common to apoptotic, rather than necrotic, cell death [17].


A putrescine-anthracene conjugate: a paradigm for selective drug delivery.

Palmer AJ, Ghani RA, Kaur N, Phanstiel O, Wallace HM - Biochem. J. (2009)

HL-60 viable cell number in the presence of Ant 4HL-60 cells were seeded at 6.8×104 cells/ml in 3.5-cm plates and grown for up to 144 h. After 48 h, cells were treated with vehicle (■) or 15 μM Ant 4 (▲). Duplicate plates were harvested at 0 h and then every 24 h. Viable cell number and cell viability were determined by Trypan Blue exclusion and microscopy. Protein content was determined uising the Lowry assay. Results are the means±S.E.M. (n=4) with two replicates per experiment. Results were analysed statistically using ANOVA with Bonferroni's multiple comparison post-hoc test (*P<0.01; **P< 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805923&req=5

Figure 2: HL-60 viable cell number in the presence of Ant 4HL-60 cells were seeded at 6.8×104 cells/ml in 3.5-cm plates and grown for up to 144 h. After 48 h, cells were treated with vehicle (■) or 15 μM Ant 4 (▲). Duplicate plates were harvested at 0 h and then every 24 h. Viable cell number and cell viability were determined by Trypan Blue exclusion and microscopy. Protein content was determined uising the Lowry assay. Results are the means±S.E.M. (n=4) with two replicates per experiment. Results were analysed statistically using ANOVA with Bonferroni's multiple comparison post-hoc test (*P<0.01; **P< 0.001).
Mentions: The IC50 value, at which 50% inhibition of cell growth was observed, was calculated to be 20 μM after 48 h exposure. Multiple studies from our laboratory have shown that IC50 values calculated from MTT curves give an overestimate of the value (F.R. Saunders and H.M. Wallace, unpublished work). Therefore in order to examine the response to Ant 4 over time in greater detail, a concentration of 15 μM was chosen (~IC30 value). At this concentration, inhibition of growth was observed by decreases in both cell number (Figure 2) and protein content (result not shown). Cytotoxic effects were observed after 24 h exposure to Ant 4. Despite the low cell numbers at longer times, cell viability remained high (86.5%), a feature common to apoptotic, rather than necrotic, cell death [17].

Bottom Line: Apoptosis was the predominant type of cell death, with mechanistic studies revealing that oxidative stress and DNA damage may have a part to play.For the first time, uptake of Ant 4 via the PTS was demonstrated both directly and indirectly in human cell lines.In addition, Ant 4 significantly reduced putrescine uptake, demonstrating that this conjugate not only used the PTS, but also could successfully compete with its native polyamine for uptake.

View Article: PubMed Central - PubMed

Affiliation: Divison of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen AB25 2ZD, UK.

ABSTRACT
Increased polyamine concentrations play an important role in the development of cancer at all stages, from initiation through to maintenance of the transformed phenotype. One way cancer cells accumulate increased concentrations of polyamines is by increased uptake of preformed polyamines via their PTS (polyamine transport system). The PTS is promiscuous and will transport a range of polyamine-based molecules. Therefore it may be that cytotoxic drugs could be attached to polyamine vectors and targeted selectively to cancer cells by utilizing the PTS. The aim of the present study was to investigate the potential of Ant 4, a putrescine-anthracene conjugate, to target cytotoxic agents to human cancer cells as a paradigm for a novel method of selective drug delivery. Ant 4 induced cytotoxicity after only 24 h exposure. Apoptosis was the predominant type of cell death, with mechanistic studies revealing that oxidative stress and DNA damage may have a part to play. For the first time, uptake of Ant 4 via the PTS was demonstrated both directly and indirectly in human cell lines. In addition, Ant 4 significantly reduced putrescine uptake, demonstrating that this conjugate not only used the PTS, but also could successfully compete with its native polyamine for uptake. However, the most interesting finding was the intracellular depletion of the polyamine pools, providing an additional mode of toxicity for Ant 4 and the possibility that this molecule may act as a 'double-edged sword': preventing cell growth by delivery of the toxic moiety and by depletion of intracellular polyamine content.

Show MeSH
Related in: MedlinePlus